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Preparation of Outer Membrane Vesicles from Myxococcus xanthus
从黄色黏球菌制备外膜囊泡   

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Abstract

Outer membrane vesicles (OMVs) represent a unique sub-cellular compartment of bacteria that may act as a scaffold for various extracellular activities, including intercellular signaling. Myxococcus xanthus (M. xanthus) is a predatory bacterium that engages in cell-cell behaviors such as fruiting body formation and contact dependent lysis of other microbes. The OMVs of M. xanthus have been shown to have an elaborate architecture of chains and tubes that can connect cells within a biofilm. These higher order OMV structures have been shown to contain proteins exchanged for community behaviors and small molecules that have antibiotic activities, and may help facilitate directed exchange. M. xanthus OMVs allow material transfer between neighboring cells for motility and predation.

Keywords: EPS(EPS), Biofilm(生物膜), Cell fractioning(细胞分级分离), Proteomics(蛋白质组学研究), Extracellular(细胞外)

Materials and Reagents

  1. 50 ml plastic tubes for handling harvested cultures (VWR International, catalog number: 62406-200 )
  2. 0.45 μm syringe filters (VWR International, catalog number: 28145-477 )
  3. 0.22 μm syringe filters (VWR International, catalog number: 28144-050 )
  4. 30 ml syringes (VWR International, catalog number: 66064-760 )
  5. 1.5 ml Eppendorf tubes (VWR International, catalog number: 89213-152 )
  6. 200 mesh formvar coated TEM grids (Electron Microscopy Sciences, catalog number: EMS200-Cu )
  7. NuncTM MicroWellTM 96-Well Optical-Bottom Plates with Coverglass Base (Thermo Fisher Scientific)
  8. Myxococcus xanthus wild type strain DZ2 (UC Regents, Berkeley)
  9. Phosphate buffered saline (PBS) (VWR International, catalog number: 97064-158 )
  10. Fluorescent lipid dye FM 4-64 (Life Technologies, Molecular Probes®, catalog number: T-13320 )
    Note: Currently, it is “Thermo Fisher Scientific, Molecular ProbesTM, catalog number: T-13320”.
  11. Uranyl acetate (Electron Microscopy Sciences)
  12. 10 mM MOPS (pH 7.6)
  13. 2 mM MgSO4
  14. 10% (w/w) Bacto casitone
  15. 5% (w/w) Bacto yeast extract
  16. CYE Media (see Recipes)

Equipment

  1. Sterile, side arm 250 ml Erlenmeyer Flasks
  2. Shaking incubator (e.g., Thermo Fisher Scientific, model: MaxQ4000 )
  3. Vortexer
  4. Centrifuge with capacity for 25 ml cultures, 5,000 x g (Refrigeration not required)
  5. Ultra-centrifuge with capacity for 1 ml samples, 140, 000 x g required and refrigeration required. (e.g., GMI, Beckman Coulter, model: L8-70M )
  6. Ultra-centrifuge tubes (fit to machine specifications)
  7. -80 °C freezer
  8. Fluorescence plate reader (e.g., Tecan Trading AG, model: Vission-100 )
  9. Transmission Electron Microscope (Philips/FEI, model: 5350 NE Dawson Creek Drive ), capable of imaging negatively-stained samples at voltages ranging between 80 kv and 200 kV equipped with a 2 k x 2 k CCD camera
  10. Autoclave for sterilizing media and flasks (or access to sterile media and growth vessels).

Software

  1. Digital Micrograph software and 2 k x 2 k CCD camera (Gatan Inc.)

Procedure

  1. In order to obtain a cell free preparation of outer membrane vesicles and vesicle chains, M. xanthus strain DZ2 (wild type) cells were harvested from restreaks on CYE Petri plates, 3-21 days old and inoculated into 250 ml Erlenmeyer flasks with 25 ml of liquid CYE media (10:1 volume of flask:volume of culture ratio should be maintained for aerobic cultures of M. xanthus). A single 25 ml culture is sufficient for isolating ~200 μg of OMVs for further analysis.
  2. Cultures were grown with shaking at 200 rpm, 32 °C for 2 days to an OD600 = 2.0 (~2 x 109 cells/ml) in a Thermo Scientific MaxQ4000 shaking incubator.
  3. Cultures were transferred to 50 ml centrifuge tubes and vortexed for 30 sec to disperse aggregates and release vesicles. Note that for M. xanthus, large cell aggregates are common, and this duration of vortexing may not be needed when adapting to other bacterial species. Cultures should be handled at room temperature for steps 3-6, as cold temperature disrupts cell viability and impinges on fractionation.
  4. The vortexed culture was centrifuged for 10 min at 5,000 x g to pellet whole cells.
  5. The supernatant was filtered through a 0.45 μm syringe filter to remove any remaining cells.
  6. Filtrate was further centrifuged as in step 4, then passed through 0.22 μm syringe filters to remove debris.
  7. Cell free filtrate was then subjected to 140,000 x g centrifugation at 4 °C using an ultracentrifuge for 1 h to harvest outer membrane vesicles, vesicle chains and membrane tubes, that will be in the resulting pellet and resuspended in 1 ml of PBS.
  8. Samples were then saved at -80 °C or subjected immediately to analysis. Samples were analyzed by two methods described below to (a) monitor the purity of vesicle fractions by fluorescent lipid dye binding and (b) monitor the purity and quality of vesicle fractions by electron microscopy. Additional analyses can also be performed, for instance, proteomic analyses have indicated that the OMV protein fraction is consistent with little to no contamination from any other large protein complexes.
    1. Sample aliquots were mixed with fluorescent lipid dye FM 4-64 (purchased as a 100 μg dry stock, stored at -20 °C until use) to a final volume of 100 μl and final concentration of 16 μM. The relative concentration of vesicle samples was determined using black 96 well plates and a fluorescence plate reader by exciting at 515 nm, measuring emission at 640 nm. Five independent samples were analyzed for each biological preparation, as mixtures are heterogeneous. 16 μM FM 4-64 in PBS should be used as a negative control, while whole cell fractions can be utilized as positive controls. Fluorescence units may vary, but successful preparations should be 10-40 fold or higher than negative control readings. 1:1 Serial dilutions of samples may be required if signal saturation occurs. Yield increases with increasing cell density and with stable surface grown cultures (biofilms) relative to aerobic liquid cultures but ~200 μg or more is obtainable with this protocol.
    2. Negative-stained TEM samples were prepared by applying 3 μl of the OMV samples in 3 replicates to 200 mesh formvar coated TEM grids, allowing 3 min to settle and rinsing briefly in distilled water. A 2.0 % solution of Uranyl acetate was applied to cover the grid before immediately blotting off and air drying. Samples were imaged on a FEI CM200 microscope with a 2k x 2k Gatan CCD camera or on a JEOL 1200 microscope, operated at 200 kV or 120 kV, respectively, with quality judged by the presence of vesicles and vesicle chains and the absence of cells or cellular debris. For more detail on EM procedures see Remis et al. (2014a), for a video of EM procedures please see Rames et al. (2014b).

Notes

Other EM analysis methods [SEM, resin-embedded sections TEM, cryo-EM of frozen-hydrated vesicle samples (Remis et al., 2014)] are also feasible for this assessment, but negative stain TEM is the simplest technique that provides this information. Light microscopy alone, due to its limited resolution, typically fails to provide accurate assessments of vesicle purifications.


Figure 1. TEM image of M. xanthus OMVs (from Remis et al., 2014) showing isolated OMVs as well as OMV chains

Recipes

  1. CYE Media
    10 mM MOPS (pH 7.6)
    2 mM MgSO4
    10% (w/w) Bacto casitone
    5% (w/w) Bacto yeast extract
    Sterilized by autoclaving

Acknowledgments

This protocol was adapted from previous work (Berleman et al., 2014; Palsdottir et al., 2009; Remis et al., 2014). This work was Supported by Lab directed research development funds from the Office of Biological and Environmental Research of the US Department of Energy under contract number DE-AC02-05CH11231 (to Manfred Auer) and the US Department of Energy VFP program (to James E. Berleman).

References

  1. Berleman, J. E., Allen, S., Danielewicz, M. A., Remis, J. P., Gorur, A., Cunha, J., Hadi, M. Z., Zusman, D. R., Northen, T. R., Witkowska, H. E. and Auer, M. (2014). The lethal cargo of Myxococcus xanthus outer membrane vesicles. Front Microbiol 5: 474.
  2. Palsdottir, H., Remis, J. P., Schaudinn, C., O'Toole, E., Lux, R., Shi, W., McDonald, K. L., Costerton, J. W. and Auer, M. (2009). Three-dimensional macromolecular organization of cryofixed Myxococcus xanthus biofilms as revealed by electron microscopic tomography. J Bacteriol 191(7): 2077-2082. 
  3. Remis, J. P., Wei, D., Gorur, A., Zemla, M., Haraga, J., Allen, S., Witkowska, H. E., Costerton, J. W., Berleman, J. E. and Auer, M. (2014a). Bacterial social networks: structure and composition of Myxococcus xanthus outer membrane vesicle chains. Environ Microbiol 16(2): 598-610.
  4. Rames, M., Yu, Y. and Ren, G. (2014b). Optimized negative staining: a high-throughput protocol for examining small and asymmetric protein structure by electron microscopy. J Vis Exp(90): e51087. 

简介

外膜囊泡(OMV)代表细菌的独特亚细胞区室,其可以充当各种细胞外活性(包括细胞间信号传导)的支架。 (Myxococcus xanthus)是一种掠夺性细菌,其参与细胞 - 细胞行为,例如子实体形成和其他微生物的接触依赖性裂解。 M.M的OMV。 xanthus已经显示具有可以连接生物膜内的细胞的链和管的精细结构。 这些更高级的OMV结构已经显示含有交换为社区行为的蛋白质和具有抗生素活性的小分子,并且可以有助于促进定向交换。 M。 xanthus OMV允许相邻细胞之间的物质转移,用于运动和捕食。

关键字:EPS, 生物膜, 细胞分级分离, 蛋白质组学研究, 细胞外

材料和试剂

  1. 50ml用于处理收获培养物的塑料管(VWR International,目录号:62406-200)
  2. 0.45μm注射器过滤器(VWR International,目录号:28145-477)
  3. 0.22μm注射器过滤器(VWR International,目录号:28144-050)
  4. 30ml注射器(VWR International,目录号:66064-760)
  5. 1.5ml Eppendorf管(VWR International,目录号:89213-152)
  6. 200目形成的TEM网格(Electron Microscopy Sciences,目录号:EMS200-Cu)
  7. Nunc?MicroWell?96孔光学底板(带盖玻璃底座)(Thermo Fisher Scientific)
  8. < em>野生型菌株DZ2(UC Regents,Berkeley)
  9. 磷酸盐缓冲盐水(PBS)(VWR International,目录号:97064-158)
  10. 荧光脂质染料FM 4-64(Life Technologies,Molecular Probes ,目录号:T-13320)
    注意:目前,"Thermo Fisher Scientific,Molecular Probes TM ,目录号: -13320 "。
  11. 乙酸乙酯(Electron Microscopy Sciences)
  12. 10mM MOPS(pH7.6)
  13. 2mM MgSO 4
  14. 10%(w/w)Bacto casitone
  15. 5%(w/w)细菌酵母提取物
  16. CYE Media(见配方)

设备

  1. 无菌侧臂250 ml锥形瓶
  2. 摇动培养箱(例如 Thermo Fisher Scientific,型号:MaxQ4000)
  3. Vortexer
  4. 离心机,容量为25ml培养物,5000×g(不需要冷藏)
  5. 超离心机,具有1ml样品的容量,140,000×g所需的并且需要冷藏。 (如 GMI,Beckman Coulter,型号:L8-70M)
  6. 超离心管(适合机器规格)
  7. -80°C冰箱
  8. 荧光板读数器(例如,Tecan Trading AG,型号:Vission-100)
  9. 透射电子显微镜(Philips/FEI,型号:5350NE Dawson Creek Drive),其能够在配备有2k×2k CCD照相机的,在80kv和200kV之间的电压下对负染色的样品成像
  10. 高压灭菌介质和烧瓶(或进入无菌培养基和生长容器)。

软件

  1. 数字显微照相软件和2k×2k CCD照相机(Gatan Inc.)

程序

  1. 为了获得外膜囊泡和囊泡链的无细胞制剂,从3-21天的CYE培养皿上的再分枝收获黄单胞菌株DZ2(野生型)细胞,并接种到带有25ml液体CYE培养基(10:1体积的烧瓶:应当维持黄色有氧培养物的培养物比例的体积)的250ml锥形瓶中。单个25ml培养物足以分离?200μg的OMV用于进一步分析
  2. 在Thermo Scientific MaxQ4000中,将培养物在200rpm,32℃下振荡生长2天至OD 600 = 2.0(?2×10 9细胞/ml)摇动培养箱。
  3. 将培养物转移至50ml离心管中并涡旋30秒以分散聚集体并释放囊泡。请注意,对于 M。 xanthus),大细胞聚集体是常见的,并且当适应其它细菌物种时,可能不需要这种持续时间的涡旋。培养物应在室温下处理3-6个步骤,因为冷温度破坏细胞活力并影响分馏。
  4. 将涡旋的培养物在5,000xg离心10分钟以沉淀全细胞。
  5. 将上清液通过0.45μm注射器过滤器过滤以除去任何剩余的细胞。
  6. 将滤液如步骤4进一步离心,然后通过0.22μm注射器过滤器以除去碎屑。
  7. 然后使用超速离心机在4℃下将无细胞滤液进行140,000×g离心1小时以收获外膜囊泡,囊泡链和膜管,其将在所得沉淀中并重悬浮于1 ml PBS。
  8. 然后将样品保存在-80℃或立即进行分析。通过下述两种方法分析样品,以(a)通过荧光脂质染料结合监测囊泡部分的纯度,和(b)通过电子显微镜监测囊泡部分的纯度和质量。还可以进行另外的分析,例如,蛋白质组分析已经表明OMV蛋白部分与来自任何其它大蛋白复合物的很少或没有污染一致。
    1. 将样品等分试样与荧光脂质染料FM 4-64混合 (作为100μg干燥原液购买,在-20℃下储存直至使用)至最终 ?体积为100μl,终浓度为16μM。相对 使用黑色96孔测定囊泡样品的浓度 板和荧光读板器通过在515nm激发,测量 发射在640nm。分析了5个独立的样品 生物制剂,作为混合物是异质的。 16μMFM 4-64 in PBS应该用作阴性对照,而全细胞级分可以 ?用作阳性对照。荧光单位可以变化,但是 成功的制剂应该是10-40倍或更高的阴性 控制读数。 1:1如果需要,可能需要连续稀释样品 信号饱和发生。产量随细胞密度的增加而增加 和相对于需氧的稳定表面生长培养物(生物膜) 液体培养物,但是约200μg或更多可用该方案获得。
    2. 阴性染色的TEM样品通过应用3μl的 OMV样品以3次重复至200网孔形式涂覆的TEM网格, 允许3分钟沉淀并在蒸馏水中短暂漂洗。一个 2.0%的乙酸铀溶液用于覆盖网格 立即吸干并风干。样品在FEI上成像 CM200显微镜用2k×2k Gatan CCD照相机或在JEOL 1200上 显微镜,分别在200kV或120kV下工作,具有质量 通过囊泡和囊泡链的存在来判断和缺乏 ?细胞或细胞碎片。关于EM过程的更多细节,参见Remis等人 ?al。(2014a),有关EM程序的视频,请参阅Rames等人(2014b)。

笔记

其他EM分析方法[SEM,树脂包埋切片TEM,冷冻水合囊泡样品的冷冻EM(Remis等人,2014)]也可用于该评估,但是负染色TEM提供此信息的最简单的技术。光学显微镜单独,由于其有限的分辨率,通常不能提供对囊泡纯化的准确评估

图1. EM的TEM图像。 xanthus OMV(来自Remis等人,2014),其显示独立的OMV和OMV链。

食谱

  1. CYE媒体
    10mM MOPS(pH7.6) 2mM MgSO 4 10%(w/w)Bacto casitone
    5%(w/w)细菌酵母提取物 高压灭菌
    灭菌

致谢

该协议改编自以前的工作(Berleman等人,2014; Palsdottir等人,2009; Remis等人,2014年) 。这项工作由美国能源部生物和环境研究办公室的实验室指导的研究开发资金支持,合同号为DE-AC02-05CH11231(给Manfred Auer)和美国能源部VFP计划(给James E. Berleman)。

参考文献

  1. 这些研究结果表明,这些研究结果表明,这些研究结果表明,这些研究结果表明, 。 黄豆球菌外膜囊泡的致命货物。 前微型生物 5:474.
  2. Palsdottir,H.,Remis,J.P.,Schaudinn,C.,O'Toole,E.,Lux,R.,Shi,W.,McDonald,K.L.,Costerton,J.W.and Auer, 三维大分子组织的冷冻固定的黄色粘球菌生物膜如电子microscopic tomography。 191(7):2077-2082。
  3. Remis,J.P.,Wei,D.,Gorur,A.,Zemla,M.,Haraga,J.,Allen,S.,Witkowska,H.E.,Costerton,J.W.,Berleman,J.E.and Auer, 细菌社会网络:黄豆粘球藻外膜囊泡链的结构和组成。 Environ Microbiol 16(2):598-610。
  4. Rames,M.,Yu,Y。和Ren,G。(2014b)。 优化的负染色:用于通过电子显微镜检查小的和不对称的蛋白质结构的高通量方案。/a> J Vis Exp (90):e51087。
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引用:Berleman, J. E., Zemla, M., Remis, J. P. and Auer, M. (2016). Preparation of Outer Membrane Vesicles from Myxococcus xanthus. Bio-protocol 6(2): e1716. DOI: 10.21769/BioProtoc.1716.
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